We have recently discovered a novel oncogenic mechanism whereby cyclin D1 bypasses CDK4/6 inhibition resulting in the stimulation of 6-phosphofructo-2-kinase (PFKFB3) and glucose metabolism. The objective of this proposal is to characterize the role of PFKFB3 in mediating adaptation or resistance to CDK4/6 inhibition and to define new approaches to increase the efficacy of CDK4/6 inhibitors in order to enable durable responses in stage IV HR+ breast cancer patients. Deregulation of the estrogen receptor (ER)-cyclin D1-CDK4/6 pathway is a hallmark of ER+ breast cancer that has prompted the development of CDK4/6 inhibitors. Although administration of CDK4/6 inhibitors to patients with ER+ advanced breast cancers have resulted in an improvement in progression free survival, almost all patients invariable develop resistance and relapse over time. Activation of glucose metabolism upon CDK4/6 inhibition is becoming increasingly evident. A key stimulator of glycolysis is the PFKFB3 enzyme which synthesizes fructose 2,6-bisphosphate (F2,6BP), a potent allosteric activator of the rate-limiting 6-phosphofructo-1-kinase (PFK1). F2,6BP controls flux throughout the entire glycolytic pathway and, as a result is required for the proliferation of cancer cells. In preliminary studies, we provide evidence that PFKFB3 expression and activity is increased in response to CDK4 inhibition in ER+ breast cancer cells and breast cancer patient tumors. Notably, we show that a PFKFB3 inhibitor, PFK-158, increases the anti-tumor activity of anti-CDK4 targeted therapy in vitro and in vivo. We postulate that PFKFB3 is an essential regulatory effector of the cyclin D1-CDK4/6 axis that serves to attenuate CDK4/6 inhibition effects by stimulating glucose metabolism. Furthermore, we hypothesize that genetic or pharmacological inhibition of PFKFB3 will increase the efficacy of CDK4/6 inhibitors by disabling the ability of the cell to sustain sufficient glucose metabolism necessary for survival and growth. Aim 1 will determine the mechanism by which CDK4/6 inhibition induces PFKFB3 expression. Aim 2 will determine the effects of combined CDK4/6 and PFKFB3 inhibition on glucose metabolism, cell cycle, growth and survival in vitro. Aim 3 will examine the metabolic and growth effects of pharmacological CDK4/6 and PFKFB3 inhibition as monotherapies and in combination in mouse models of breast cancer in vivo. We anticipate that our studies will support the clinical testing of PFKFB3 inhibitors in combination with CDK4/6 inhibitors to increase the survival of advanced ER+ breast cancer patients.